The present invention relates to nucleotide sequences that can be used to produce polypeptides that exhibit the characteristics of the nucleocapsid N protein, or one of two glycoproteins G1 and G2, synthesized by the Hantaan virus. The present invention further relates to a method of producing a Hantaan virus vaccine comprised of at least one of the N protein, G.sub.1 and G2.
Each year, hundreds and thousands of people in endemic areas, such as China and Korea, developed infection by Hantaan virus, the etiological agent of Korean hemorrhagic fever, resulting in high morbidity and mortality, not only among the natives in those areas, but also among visitors and service personnel assigned to those areas. In recent years, Hantaviruses have been isolated from rodent populations in endemic and epidemic areas, as well as here in the United States. There is no known cure for such a viral infection, it being generally allowed to run its course until terminated by death or development of natural immunity against the virus.
Because of the extremely infectious nature of the Hantaan virus, containment of this virus for purposes of study poses a special problem. For this reason, it has not been practicable to grow large quantities of live Hantaan virus in the laboratories to extract its immunogenic portions for vaccination purposes. By the same token, detailed information concerning the Hantaan virus has been limited heretofore. Although some information is available about its RNA genome, for example, there is insufficient information available to permit production of viral antigens in the laboratory without propagating the live Hantaan virus. If the nucleotide sequences encoding antigenic portions of the Hantaan virus can be identified, it would be possible to synthesize such antigens in a cell-free system or to genetically-engineer a vaccine. Furthermore, if Hantaan viral infection can be diagnosed at an early stage, for example, by using viral cDNA probes, proper patient management at such a stage may improve prognosis of the disease.
The Hantaan virus belongs to the Hantavirus genus of the Bunyaviridae family. Viruses in this family all possess tripartite, single-stranded, negative-sense RNA species, designated large (L) , medium (M) and small (S), respectively, in accordance with their respective molecular weights. Each RNA species is enclosed in its own nucleocapsid structure, but all three RNA species are surrounded by a lipid envelope containing two virus-specific glycoproteins, known as G1 and G2 glycoproteins [see Schmaljohn et al, J. Infect. Dis. 148: 1005-1101 (1983) Schmaljohn and Dalrymple, in SEGMENTED NEGATIVE STRAND VIRUSES, R. W. Compans and D. H. Bishop, eds., pp. 117-124 (Academic Press, 1984). The Hantaan virus is negative-sense in that it has to produce a positive-sense, complementary RNA ("cRNA") to act as messenger RNA ("mRNA").
Immunogenic Hantaan viral proteins have been identified by Elliott et al, J. Gen. Virol. 65: 1282-1293 (1984), who showed that sera from patients who had recovered from Hantaan virus infection precipitates three Hantaan viral proteins, a nucleocapsid N protein and G1 and G2 glycoproteins. It was not known, however, which of the three RNA species of Hantaan virus, or which segment(s) of such species, encode for each of the N, G1 and G2 proteins.
In the production of immunogenic proteins for vaccination purposes, it is not desirable to include in such vaccines other proteins or substances of unknown effect which may be harmful, e.g., pyrogenic substances. Therefore, to produce immunogenic proteins using genetic engineering techniques, i.e., in vitro techniques to produce DNA molecules containing novel combinations of genes or nucleotide sequences, it is necessary to know precisely which segment of a genome encodes the desired protein. The uncertainty with regard to which segments of the Hantaan viral genome encode the desired proteins can be more fully appreciated by considering what is known about viruses of the Bunyavirus and Phlebovirus genera, other members of the Bunyaviridae family. The Bunyaviruses S-RNA molecules utilize an overlapping reading frame strategy in its cRNA to encode a nucleocapsid N protein and a non-structural ("NS.sub.S ") protein. See Fuller et al, J. Gen. Virol. 64: 1705-1714 (1983). In other words, if the nucleotide sequence for the CRNA of S-RNA species is represented by the formula 1-2-3-4-5-6-7-8-9-10, the nucleotide sequence encoding the N protein may be represented by the sequence 1-2-3-4-5-6, while the sequence encoding for the NSS protein may be represented by 3-4-5-6-7-8. Therefore, to produce only the N protein of Bunyavirus by genetic engineering techniques, the sequence 1-2-3-4-5-6, but not 7-8, will have to be present.
On the other hand, viruses of the Phlebovirus genus utilize an ambisense coding strategy in their S-RNA: a N protein is encoded by a nucleotide sequence at the 5' end of the cRNA molecule, and a NS.sub.S protein is encoded by a nucleotide sequence at the 5' end of the viral RNA molecule. Synthesis of the N protein by genetic engineering techniques in this instance will require the presence of a nucleotide sequence comprising the 3' end of the viral RNA molecule, while synthesis of the NS.sub.S protein would require the presence of the 5' and of the viral RNA molecule.
In contrast, studies to date on viruses of the Phlebovirus and Bunyavirus genera revealed that a single long open-reading-frame (ORF) coding strategy was employed in their CRNA complementary to M-RNA to encode single gene products comprising both G1 and G2 glycoproteins. In the Bunyaviruses, the M-RNA additionally encodes for a NS.sub.M ("non-structural.sub.M ") polypeptide. In the Phleboviruses, sufficient coding information was found which could produce a NS.sub.M polypeptide, but such polypeptide has not been identified in infected cells. To obtain G1 and G2 glycoproteins but not the NS.sub.M proteins, therefore, the specific nucleotide sequence encoding G1 and G2 glycoproteins will have to be identified so that the sequence encoding for NS.sub.M protein can be excluded.
Based upon the present state of the art, it has been impossible to predict a Priori what coding strategy is utilized in the Hantaan virus, and which segments of its genome comprise the nucleotide sequences that encode the polypeptides corresponding to the immunogenic N protein, and G1 and G2 glycoproteins. In order to produce such proteins without the use of live viruses, however, this information is needed to avoid the production of undesirable proteins.